Technical Abstract:
Salmonella enteritidis (SE) is the primary pathogen responsible for Salmonella-associated food borne outbreaks, which is spread to humans mainly through contaminated eggs. Understanding the pathogenesis of SE in poultry could lead to strategies to modulate the immune responses to better control and prevent SE infections in birds, therefore leading to effective reduction or elimination of SE associated food poisoning. Previously, we have observed that SE is able to survive in chicken macrophage cell line HD 11 cells for a long time period before the clearance happens, while Salmonella typhimurium (ST) invasion results in immediate cell death of the infected HD 11 cells. By using both green fluorescence protein (GFP) engineered ST and SE, we found that ST quickly induced much higher levels of nitric oxide production in infected HD 11 cells than SE, while at later time periods, ST infected cells had less nitric oxide accumulated in culture than SE This may be due to the f loss of the majority of cells infected with ST presumably due to the induction of ST-mediated apotosis. To test this hypothesis, we used Annexin V-PE apoptosis kit and two-color flow cytometry. When compared to SE, ST induced faster and more strong apoptotic response in HD 11 cells.death. NG-monomethyl L-arginine (L-NMMA), an inhibitor for inducible nitric oxide synthase (iNOS), blocked the nitric oxide production in both ST- and SE- infected HD 11 cells by up to 50%, and almost completely prevent apoptosisalthough GFP-SE and GFP-ST remained intracellularly. This suggested that both SE and ST induced apoptosis in HD 11 cells through an iNOS-dependent pathway, which lead to theintracellular bacterial killing and induction of host immunity. However, the exact mechanism that SE use to limit or avoid iNOS-dependent apoptosis pathway and survive inside macrophages, needs to be better characterized. A synthetic CpG containing oligonuleatide sequence (ODN) and designated as HuI, was found by our group to be induce apoptosis in HD 11 cells by stimulating a large amount of nitric oxide in culture. By adding HuI into the culture, we were able to increase apoptosis and eliminate intracellular SE bacteria in HD 11 cells